Anti-tumor agents

ABSTRACT

A method for treating subjects with abnormal cell proliferation is provided. The method involves administering to subjects in need of such treatment an effective amount of an agent of Formula I, to inhibit cell proliferation such as that associated with tumor growth and metastasis. A method for inhibiting angiogenesis in an abnormal proliferative cell mass by the administration of an agent of Formula I is also provided.

RELATED APPLICATIONS

This application is a continuation of non-provisional patent applicationSer. No. 11/476,524, entitled “Anti-Tumor Agents”, filed on Jun. 28,2006 now issued as U.S. Pat. No. 7,282,484, which is a continuation ofnon-provisional patent application Ser. No. 11/131,478 entitled“Anti-Tumor Agents”, filed on May 17, 2005 and now issued as U.S. Pat.No. 7,259,138, which is a continuation of non-provisional patentapplication Ser. No. 10/384,121 entitled “Anti-Tumor Agents”, filed Mar.7, 2003 now issued as U.S. Pat. No. 6,949,514, which is a continuationof non-provisional patent application Ser. No. 09/578,363 entitled“Anti-Tumor Agents”, filed May 25, 2000 and now issued as U.S. Pat. No.6,890,904, which claims priority under 35 U.S.C. §119(e) to theprovisional patent application entitled “Anti-Tumor Agents” filed May25, 1999, and assigned Ser. No. 60/135,861, the entire contents of allof which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods for the treatment of abnormalproliferative disorders. The methods involve administering certaincompounds to inhibit proliferation and angiogenesis in an abnormalproliferative cell mass.

BACKGROUND OF THE INVENTION

Abnormal cell proliferation is usually characterized by an increasedrate of division and in some cases uncontrolled growth. One example of aproliferative cell disorder is a tumor. In addition to posing a serioushealth risk in and of themselves, primary malignant tumors areparticularly problematic given their tendency to invade surroundingtissues and metastasize to distant organs in the body. To date, the mostfrequently used methods for treating neoplasia, especially solid tumorforms of neoplasia, include surgical procedures, radiation therapy, drugtherapies, and combinations of the foregoing. These methods involvesignificant risk (e.g., of infection, death) to the patient. Moreimportantly, the probability of eliminating all malignant cells is smallparticularly if the zone of malignant growth is not well defined or ifthe primary tumor has metastasized by the time of surgery. Achievingtherapeutic doses effective for treating the cancer is often limited bythe toxic side effects of the anti-cancer agent on normal, healthytissue. An ideal anti-cancer agent has tissue specificity, therebyreducing side-effects on normal (dividing) cells.

Recently, a model of anti-cancer therapy has been proposed and validatedthat targets the vasculature of solid tumors rather than the malignantcells themselves. Most, if not all, solid tumors require a blood supplysystem for oxygenation, nutrient delivery and waste product removal. Theneed for neovascularization is particularly acute if the tumor is togrow beyond the confines of the normal blood supply system. As a result,tumors which do attain a certain size are able to elicit the growth ofnew blood vessels from the surrounding endothelial cells, through aprocess called angiogenesis, through the release of angiogenic factors.

In view of the foregoing, a need exists to identify agents for treatingcancer and metastasis.

SUMMARY OF THE INVENTION

The invention solves these and other problems by providing methods andrelated compositions for treating conditions characterized by abnormalcell proliferation, including, but not limited to, cancer andmetastasis. The invention is based, in part, on the observation thatcompounds of Formula I are able to inhibit the enzymatic activity offibroblast activation protein-alpha (FAP-α).

In one aspect, the invention provides a method for treating a subjecthaving a condition characterized by abnormal mammalian cellproliferation. The method comprises administering to a subject in needof such treatment, an agent in an amount effective to inhibit theproliferation, wherein the agent is a compound of Formula I:

Formula I PR

wherein P is a targeting group which binds to the reactive site of FAP-αor other post proline-cleaving enzyme and can be a peptide or apeptidomimetic, and wherein R is a reactive group capable of reactingwith a functional group in FAP-α or other post proline cleaving enzyme,preferably in the reactive site of FAP-α or other post proline cleavingenzyme. The reactive compound may be selected from the group consistingof organo boronates, organo phosphonates, fluoroalkylketones,alphaketos, N-peptiolyl-O-(acylhydroxylamines), azapeptides, azetidines,fluoroolefins dipeptide isoesteres, peptidyl (alpha-aminoalkyl)phosphonate esters, aminoacyl pyrrolidine-2-nitriles and4-cyanothiazolidides.

One group of Formula I compounds useful in the invention can be furtherdefined by Formula II

wherein m is an integer between 0 and 10, inclusive; A and A₁ may be L-or D-amino acid residues such that each A in A_(m) (i.e., where m>1) maybe a different amino acid residue from every other A in A_(m); the Cbonded to B is in the L-configuration; the bond between A₁ and N and, insome embodiments, between A₁ and A_(m) are peptide bonds; and each X₁and X₂ is, independently, a hydroxyl group or a group capable of beinghydrolyzed to a hydroxyl group in aqueous solution at physiological pH.By “the C bonded to B is in the L-configuration” is meant that theabsolute configuration of the C is like that of an L-amino acid. Thus,the

group has the same relationship to the C as the —COOH group of anL-amino acid has to its α carbon. In some embodiments, A and A₁ areindependently proline or alanine residues. In some embodiments, m is 0.In some embodiments, X₁ and X₂ are hydroxyl groups. In some embodiments,the inhibitor is L-Ala-L-boroPro. In still other embodiments, inhibitoris L-Pro-L-boroPro.

In addition to agents of Formula II, other agents useful in theinvention include those in which the proline residue in Formula II isreplaced with another amino acid residue such as, for example, lysine,alanine or glycine. As well, derivatives of Formula II in which theboronate group is replaced with a reactive group as described above arealso useful in the invention. In preferred embodiments, the agent isVal-boro-Pro.

Some representative agents of Formula I can be further defined byFormula III as follows:

wherein m is an integer between 0 and 10, inclusive; A and A₁ areL-amino acid residues (for glycine there is no such distinction) suchthat the A in each repeating bracketed unit can be a different aminoacid residue; the C bonded to B is in the L-configuration; the bondsbetween A and N, A₁ and C, and between A₁ and N are peptide bonds; andeach X₁ and X₂ is, independently, a hydroxyl group or a group capable ofbeing hydrolyzed to a hydroxyl group in aqueous solution atphysiological pH. By “the C bonded to B is in the L-configuration” ismeant that the absolute configuration of the C is like that of anL-amino acid.

In general, naturally occurring compounds which contain a chiral centerare only in one stereo isomeric form, either D or L. The naturallyoccurring amino acids are the L stereo isomers; however, the inventionembraces amino acids which can be in the D stereo isomer configuration.

In one aspect, the agent of Formula I is administered to a subject inneed thereof in an amount effective to inhibit abnormal mammalian cellproliferation. The subjects to be treated are subjects having acondition characterized by abnormal mammalian cell proliferation. Incertain embodiments, the subjects preferably are otherwise free ofsymptoms calling for hemopoietic stimulation and, in particular, arefree of symptoms calling for treatment with a compound for stimulatingan immune response. In some embodiments, the subjects to be treated donot exhibit symptoms requiring hemopoietic stimulation and have normalor protective levels of hemopoietic cells. The subject to be treated mayhave normal hemopoietic activity. Included are subjects who are HIVpositive but who have normal hemopoietic activity. In anotherembodiment, the subject is HIV negative. In certain embodiments, thesubjects are not myeloid or lymphoid suppressed or are not candidatesfor treatment with an agent which causes such suppression at the time oftreatment with the methods of the invention.

The invention is further premised, in part, on the discovery thatcompounds of Formula I (e.g., Val-boro-Pro) have anti-tumor activity inmelanoma and fibrosarcoma.

Thus, in another aspect, the subjects are treated with the agent ofFormula I in a manner and in an amount so as to inhibit proliferation ofa primary tumor, or to inhibit metastatic spread or growth whileminimizing the potential for systemic toxicity. In certain embodiments,the abnormal mammalian cell proliferation is manifested as a tumor. Someconditions intended to be treated by the method of the invention includebenign (i.e., non-cancerous), pre-malignant and malignant (i.e.,cancerous) tumors. In some embodiments, the condition characterized byabnormal mammalian cell proliferation is further characterized by thepresence of reactive stromal fibroblasts.

In other embodiments, the abnormal mammalian cell proliferation isselected from the group consisting of a carcinoma, a sarcoma, and amelanoma. In yet other embodiments, the condition is selected from thegroup consisting of breast cancer, colorectal cancer, ovarian cancer,prostate cancer, pancreatic cancer, kidney cancer, lung cancer, melanomaand fibrosarcoma. In still other embodiments, the condition is selectedfrom the group consisting of bone and connective tissue sarcomas,examples of which include, but are not limited to, osteosarcoma andfibrosarcoma.

In still other embodiments, the abnormal mammalian cell proliferation isin epithelial cells, meaning that it is epithelial cells which areabnormally proliferating. Some conditions characterized by abnormalmammalian epithelial cell proliferation include adenomas of epithelialtissues such as the breast, colon and prostate, as well as malignanttumors. According to other embodiments of the invention, a method isprovided for treating a subject having a metastasis of epithelialorigin.

According to some embodiments of the invention, the agent isadministered locally. In some embodiments, the agent is targeted to atumor. This can be achieved by the particular mode of administration.For example, easily accessible tumors such as breast or prostate tumorsmay be targeted by direct needle injection to the site of the lesion.Lung tumors may be targeted by the use of inhalation as a route ofadministration.

In some embodiments, the agents may be administered in a systemicmaimer, via administration routes such as, but not limited to, oral,intravenous, intramuscular and intraperitoneal administration. Systemicadministration routes may be preferred, for example, if the subject hasmetastatic lesions. In other embodiments, the agent is administered in asustained release formulation.

In administering the compounds of the invention to subjects, dosingamounts, dosing schedules, routes of administration and the like may beselected so as to affect the other known activities of these compounds.For example, amounts, dosing schedules and routes of administration canbe selected as described herein, whereby therapeutically effectivelevels for inhibiting proliferation are provided, yet therapeuticallyeffective levels for restoring hemopoietic deficiency are not provided.

In addition, agents can be selected that are effective asanti-proliferative agents or as anti-angiogenic agents but arerelatively ineffective as hemopoietic cell stimulatory or activatingagents. Thus, certain subjects who require both hemopoietic stimulationand/or activation and proliferation and/or angiogenic inhibition may betreated with different agents of the invention simultaneously, one eachfor the desired therapeutic effect, or with a single compound but indifferent dosages, schedules, and/or route to achieve both hemopoieticstimulation and proliferation inhibition at therapeutic levels.

In some embodiments of the invention, a method is provided in which theagent is administered in combination with an anti-proliferativecompound, such as an anti-cancer compound. In another embodiment, theagent is administered in combination with surgery to remove an abnormalproliferative cell mass. In a related embodiment, the agent isadministered to a patient who has had surgery to remove an abnormalproliferative cell mass.

In another aspect, the invention provides a method for inhibitingangiogenesis in a subject having a condition characterized by abnormalmammalian cell proliferation comprising administering to a subject inneed of such treatment, an agent in an amount effective to inhibitangiogenesis in an abnormal proliferative cell mass, wherein the agentis a compound of Formula I.

In some embodiments, the abnormal mammalian cell proliferation ismanifested as a tumor. In another embodiment, the abnormal mammaliancell proliferation is selected from the group consisting of a carcinoma,a sarcoma, and a melanoma. In still another embodiment, the conditioncharacterized by abnormal mammalian cell proliferation is a metastasis.In other embodiments, the condition is selected from the groupconsisting of breast cancer, colorectal cancer, ovarian cancer, prostatecancer, pancreatic cancer, kidney cancer, lung cancer, melanoma andfibrosarcoma. In another embodiment, the abnormal mammalian cellproliferation is in epithelial cells, meaning that epithelial cells areabnormally proliferating.

In one embodiment, the agent is administered locally. In anotherembodiment, the agent is targeted to a tumor. In another embodiment, theagent is administered in a sustained release formulation. In yet anotherembodiment, the agent is administered systemically.

In some embodiments, the agent is administered in combination withsurgery to remove an abnormal proliferative cell mass. In otherembodiments, the agent is administered to a patient who has had surgeryto remove an abnormal proliferative cell mass.

In one embodiment, the agent is administered in combination with ananti-proliferative compound such as an anti-cancer compound. In otherembodiments, the agent is administered in combination with ananti-angiogenic compound. In yet other embodiments, the agent isadministered with an anti-cancer compound and an anti-angiogeniccompound.

In one embodiment, the subject has normal hemopoietic activity. Inanother embodiment, the subject is HIV negative.

According to still another aspect of the invention, pharmaceuticalpreparations are provided that comprise an agent of Formula I, asdescribed above, and a pharmaceutically-acceptable carrier. Formula Iagents are present in the pharmaceutical preparations in an amounteffective for inhibiting proliferation in primary or secondary (e.g.,metastatic) malignant lesions. These pharmaceutical preparations maycontain the agent of Formula I alone or in combination with othercompounds (e.g., anti-cancer compounds and/or anti-angiogeniccompounds).

In one aspect, a pharmaceutical preparation is provided comprising anagent of Formula I, at least one other anti-cancer compound (i.e., ananti-cancer compound other than an agent of Formula I), and apharmaceutically acceptable carrier. In another aspect, a pharmaceuticalpreparation is provided which comprises an agent of Formula I, at leastone other anti-angiogenic compound (i.e., an anti-angiogenic compoundother than an agent of Formula I), and a pharmaceutically acceptablecarrier.

In other embodiments, anti-cancer cocktails containing the agent of theinvention and other anti-proliferative compounds and/or otheranti-angiogenic compounds as described herein are also provided. Instill other embodiments, the agents of Formula I are used in thepreparation of a medicament for treating subjects having conditionscharacterized by abnormal mammalian cell proliferation.

In still other embodiments, the agent may be targeted to a cell mass(e.g., a tumor) through the use of a targeting compound specific for aparticular tissue or tumor type. In some embodiments, the agents of theinvention may be targeted to primary or in some instances, secondary(i.e., metastatic) lesions through the use of targeting compounds whichpreferentially recognize a cell surface marker.

These and other aspects of the invention will be described in greaterdetail below. Throughout this disclosure, all technical and scientificterms have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains unless definedotherwise.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a is a comparison of response of Fischer D⁺ rat and BM stromalcells to PT-100 in vitro by measurement of interleukin-6 (IL-6) release.

FIG. 1 b is a comparison of response of Fischer D⁻ rat and BM stromalcells to PT-100 in vitro by measurement of interleukin-1 (IL-6) release.

FIG. 2 is a profile of FAP-α and CD26 cell surface expression by humanbone marrow stromal cells.

FIG. 3 a is the PT-100 dose response of FAP-α⁺ CD26⁻ primary humanstromal cells by measurement of granulocyte-colony stimulating factor(G-CSF) release.

FIG. 3 b is the PT-100 dose response of FAP-α⁺ CD26⁻ primary humanstromal cells by measurement of inhibition of DPPIV-like activity.

FIG. 4 is a bar graph showing the effect of PT-100 treatment onestablished subcutaneous B16-F10 tumors.

FIG. 5 a is a bar graph showing the effect of PT-100 on WEHIsubcutaneous growth (in terms of tumor volume) in vivo on day 20 postinjection.

FIG. 5 b is a bar graph showing the effect of PT-100 on WEHIsubcutaneous growth (in terms of tumor weight) in vivo on day 20 postinjection.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to the inhibition of cellular proliferationand angiogenesis in conditions characterized by abnormal, unwantedmammalian cell proliferation. The invention is useful, inter alia, inthe treatment of proliferative disorders including benign, pre-malignantand malignant tumors. In certain embodiments, the methods are directedtowards the treatment of subjects with carcinoma or metastatic lesionsof epithelial origin. The invention is based, in part, on the findingthat PT-100, the dipeptide valine-proline-boronic acid, i.e.,(ValboroPro), is able to inhibit fibroblast activation protein (FAP-α),and also exhibits anti-tumor activity against cancers such as, forexample, melanoma and fibrosarcoma.

According to one aspect of the invention, a method is provided fortreating a subject having a condition characterized by an abnormalmammalian cell proliferation, using compounds of Formula I:

PR   Formula I

wherein P is a targeting group which binds to the reactive site of FAP-αor other post proline-cleaving enzyme, and R is a reactive group capableof reacting with a functional group in the reactive site of FAP-α orother post proline-cleaving enzyme. Post proline-cleaving enzymes areenzymes which have a specificity for removing Xaa-Pro (where Xaarepresents any amino acid) dipeptides from the amino terminus ofpolypeptides. Examples of post-proline cleaving enzymes include, but arenot limited to, CD26, dipeptidyl peptidase IV (DP IV) and dipeptidylaminopeptidase IV.

The P targeting group can be composed of single or multiple residues ofpeptide or peptidomimetic nature, provided that such residues do notinterfere significantly, and most preferably improve the site-specificrecognition of FAP-α or other post proline-cleaving enzyme by the agentof Formula I. In certain embodiments, the portion of the P targetinggroup that is involved in binding to the reactive site of FAP-α or otherpost proline-cleaving enzyme is formed of amino acids and the remainingportion of P is formed of non-amino acid components. According to theparticular embodiment, P can be composed wholly of amino acid residues,wholly of non-amino acid substituents, or a combination of both.

In general, the targeting group (i.e., P) is covalently coupled to thereactive group. In some embodiments, the covalent coupling occurs via acarboxyl group at the carboxyl terminal amino acid in the P group. Incertain embodiments, P may be 30, 20, 10 or less than 10 residues inlength.

The development of phage display libraries and chemical combinatoriallibraries from which synthetic compounds can be selected which mimic thesubstrate of a protease permits the identification of further targetinggroups to which an R group can be covalently attached to form a bindingmoiety which binds or associates with the reactive site of the proteaseand which forms a complex with a functional group in the proteasereactive site. Such libraries can be screened to identify non-naturallyoccurring putative targeting groups by assaying protease cleavageactivity in the presence and absence of the putative phage displaylibrary molecule or combinatorial library molecule and determiningwhether the molecule inhibits cleavage by the protease of a knownsubstrate or of a substrate analog (e.g., a chromophoric substrateanalog which is easily detectable in a spectrophotometric assay). Thosephage library and/or combinatorial library molecules which exhibitinhibition of for example, FAP-α, then can be covalently coupled to thereactive groups disclosed herein and again tested to determine whetherthese novel molecules selectively bind to, for example, FAP-α (e.g., byrepeating the above-noted screening assay). In this manner, a simple,high-through-put screening assay is provided for identifyingnon-naturally occurring targeting groups of the invention.

P targeting groups can be synthesized from peptides or otherbiomolecules including but not limited to saccharides, fatty acids,sterols, isoprenoids, purines, pyrimidines, derivatives or structuralanalogs of the above, or combinations thereof and the like. Alsoenvisioned in the invention is the use of targeting groups made frompeptoids, random bio-oligomers (U.S. Pat. No 5,650,489),benzodiazepines, diversomeres such as dydantoins, benzodiazepines anddipeptides, nonpeptidal peptidomimetics with a beta-D-glucosescaffolding, oligocarbamates or peptidyl phosphonates. Many, if not all,of these compounds can be synthesized using recombinant or chemicallibrary approaches. A vast array of candidate targeting groups can begenerated from libraries of synthetic or natural compounds. The methodsof the invention utilize this library technology to identify smallpeptides which bind to protease reactive sites. One advantage of usinglibraries for inhibitor identification is the facile manipulation ofmillions of different putative candidates of small size in smallreaction volumes (i.e., in synthesis and screening reactions). Anotheradvantage of libraries is the ability to synthesize targeting groupswhich might not otherwise be attainable using naturally occurringsources, particularly in the case of non-peptide moieties.

Examples of reactive groups useful in the invention include organoboronates, organo phosphonates, fluoroalkylketones, alphaketos,N-peptiolyl-O-(acylhydroxylamines), azapeptides, azetidines,fluoroolefins dipeptide isoesteres, peptidyl (alpha-aminoalkyl)phosphonate esters, aminoacyl pyrrolidine-2-nitriles and4-cyanothiazolidides.

It is to be understood that the terms ‘agent’ or ‘agent of theinvention’ or ‘agent of Formula I’ are used interchangeably to mean allof the derivatives of Formula I described herein.

Some representative agents of Formula I can be further defined byFormula II as follows:

wherein m is an integer between 0 and 10, inclusive; A and A₁ may be L-or D-amino acid residues (for glycine there is no such distinction) suchthat each A in A_(m) may be an amino acid residue different from anotheror all other A in A_(m); the C bonded to B is in the L-configuration;the bond between A₁ and N and, in some embodiments, the bond between Aand A₁ are peptide bonds; and each X₁ and X₂ is, independently, ahydroxyl group or a group capable of being hydrolyzed to a hydroxylgroup in aqueous solution at physiological pH. By “the C bonded to B isin the L-configuration” is meant that the absolute configuration of theC is like that of an L-amino acid. Thus, the

group has the same relationship to the C as the —COOH group of anL-amino acid has to its α carbon. In various embodiments, A and A₁ areindependently proline or alanine residues; m is 0; X₁ and X₂ arehydroxyl groups; the inhibitor is L-Ala-L-boroPro; and the inhibitor isL-Pro-L-boroPro.

Other agents useful in the methods and compositions of the invention arederivatives of Formula II in which each and every A in A_(m) mayindependently be a non-amino acid residue. Thus, the plurality of A(i.e., A_(m), wherein m>1) may be a peptide or a peptidomimetic whichmay include, in whole or in part, non-amino acid residues such assaccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines,derivatives or structural analogs of the above, or combinations thereofand the like. The plurality of A in A_(m) may also be comprised of acombination of amino acid and non-amino acid residues. It also ispossible to substitute non-naturally occurring amino acids, such as2-azetidinecarboxylic acid or pipecolic acid (which have 6-membered, and4-membered ring structures respectively) for the proline residue.Representative structures of transition-state analog-based inhibitorsXaa-boroPro of Formula II, include Lys-BoroPro, Pro-BoroPro andAla-BoroPro in which “boroPro” refers to the analog of proline in whichthe carboxylate group (COOH) is replaced with a boronyl group [B(OH)₂].Alternative compounds of the invention have an analogous structure inwhich the boronyl group is replaced by, for example, a phosphonate or afluoroalkylketone, alphaketos, N-peptiolyl-O-(acylhydroxylamines),azapeptides, azetidines, fluoroolefins dipeptide isoesteres, peptidyl(alpha-aminoalkyl) phosphonate esters, aminoacyl pyrrolidine-2-nitrilesand 4-cyanothiazolidides. It is to be understood that each and everyreactive group described herein can be substituted for the reactivegroup of Formula II (i.e., boronyl group).

All amino acids, with the exception of glycine, contain an asymmetric orchiral carbon and may contain more than one chiral carbon atom. Theasymmetric α carbon atom of the amino acid is referred to as a chiralcenter and can occur in two different isomeric forms. These forms areidentical in all chemical and physical properties with one exception,the direction in which they can cause the rotation of plane-polarizedlight. These amino acids are referred to as being “optically active,”i.e., the amino acids can rotate the plane-polarized light in onedirection or the other.

The four different substituent groups attached to the α carbon canoccupy two different arrangements in space. These arrangements are notsuperimposable mirror images of each other and are referred to asoptical isomers, enantiomers, or stereo isomers. A solution of onestereo isomer of a given amino acid will rotate plane polarized light tothe left and is called the levorotatory isomer [designated (−)]; theother stereo isomer for the amino acid will rotate plane polarized lightto the same extent but to the right and is called dextrorotatory isomer[designated (+)].

A more systematic method for classifying and naming stereo isomers isthe absolute configuration of the four different substituents in thetetrahedron around the asymmetric carbon atom (e.g., the α carbon atom).To establish this system, a reference compound was selected(glyceraldehyde), which is the smallest sugar to have an asymmetriccarbon atom. By convention in the art, the two stereo isomers ofglyceraldehyde are designated L and D. Their absolute configurationshave been established by x-ray analysis. The designations, L and D, alsohave been assigned to the amino acids by reference to the absoluteconfiguration of glyceraldehyde. Thus, the stereo isomers of chiralcompounds having a configuration related to that of L-glyceraldehyde aredesigned L, and the stereo isomers having a configuration related toD-glyceraldehyde are designated D, regardless of the direction in whichthey rotate the plane-polarized light. Thus, the symbols, L and D, referto the absolute configuration of the four substituents around the chiralcarbon.

In general, naturally occurring compounds which contain a chiral centerare only in one stereo isomeric form, either D or L. The naturallyoccurring amino acids are the L stereo isomers; however, the inventionembraces amino acids which can be in the D stereo isomer configuration.

Most amino acids that are found in proteins can be unambiguously namedusing the D L system. However, compounds which have two or more chiralcenters may be in 2^(n) possible stereo isomer configurations, where nis the number of chiral centers. These stereo isomers sometimes aredesignated using the RS system to more clearly specify theconfigurations of amino acids that contain two or more chiral centers.For example, compounds such as threonine isoleucine contain twoasymmetric carbon atoms and therefore have four stereo isomerconfigurations. The isomers of compounds having two chiral centers areknown as diastereomers. A complete discussion of the RS system ofdesignating optical isomers for amino acids is provided in Principles inBiochemistry, editor A. L. Lehninger, page 99-100, supra. A briefsummary of this system follows.

The RS system was invented to avoid ambiguities when a compound containstwo or more chiral centers. In general, the system is designed to rankthe four different substituent atoms around an asymmetric carbon atom inorder of decreasing atomic number or in order of decreasing valancedensity when the smallest or lowest-rank group is pointing directly awayfrom the viewer. The different rankings are well known in the art andare described on page 99 of Lehninger (supra). If the decreasing rankorder is seen to be clock-wise, the configuration around the chiralcenter is referred to as R; if the decreasing rank order iscounter-clockwise, the configuration is referred to as S. Each chiralcenter is named accordingly using this system. Applying this system tothreonine, one skilled in the art would determine that the designation,L-threonine, refers to (2S, 3R)-threonine in the RS system. The moretraditional designations of L-, D-, L-allo, and D-allo, for threoninehave been in common use for some time and continue to be used by thoseof skill in this art. However, the R S system increasingly is used todesignate the amino acids, particularly those which contain more thanone chiral center.

Many of the agents of the invention and methods for their manufacturehave been previously disclosed in U.S. Pat. No. 4,935,493, the contentsof which are incorporated by reference herein.

The invention features compounds having the structure

and salts thereof, where T is a boronate group of the formula

where each D¹ and D² independently, is a hydroxyl group or a group whichis capable of being hydrolyzed to a hydroxyl group in aqueous solutionat physiological pH; or T is a group of the formula

where G is either H, F or an alkyl group containing 1 to about 20 carbonatoms and optional heteroatoms which can be N, S, or O; or T is aphosphonate group of the formula

where each J, independently, is O-alkyl, N-alkyl, or alkyl (eachcontaining about 1-20 carbon atoms) and, optionally, heteroatoms whichcan be N, S, or O; where T is a group able to form a complex with thecatalytic site of an enzyme; X includes one or more amino acids,

Y is

and each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, independently, is a groupwhich does not interfere significantly (i.e., does not raise the Ki ofthe compound to greater than 10⁻⁵M) with site-specific recognition ofthe compound by the enzyme, while permitting a complex to be formedbetween the compound and the enzyme.

In preferred embodiments, T is a boronate group, each D¹ and D²,independently, is OH or F or D¹ and D² together form a ring containing 1to about 20 carbon atoms, and optionally heteroatoms which can be N, S,or O; each R¹⁻⁸ is H; X mimics the substrate recognized by the enzyme,for example X is pro-, thr-pro-, ala-pro-, ala-ala-pro-, ser-thr-pro-,pro-ser-, pro-thr- or ser-pro- (pro=proline, thr=threonine, andser=serine); X contains both an amino acid and a blocking group, such asan acetyl group; the enzymes inhibited by the compounds of the inventionare post prolyl cleaving enzymes, most preferably serine proteases, evenmore preferably IgAl proteases; and the analog has a binding constant ofat least 10⁻⁷M, most preferably 10⁻¹⁰M.

The compounds of the invention can be designed so that they areresistant to attack by agents which might otherwise cause theircatabolic degradation by cleavage of one or more peptide bonds in thepeptides. For example, an N-terminal blocking group, such as acetyl, canincrease the half-life of the compounds in vivo, and thus improvesinhibition.

The structure of such blocking groups can vary widely. In one blockingreaction, a hydrogen atom of the amino terminal amino group is replaced,generally in a dehydration reaction. Thus, blocking groups such as

are readily added to a peptide chain. Others include

Such N-terminal blocking groups may be employed not only to protectamino-terminal groups, but also may protect side chain amino group ofamino acids which make up X, such as where X includes Lys or Arg.Similarly, amino acid residues having acidic or hydroxy side chains canbe protected in the form of t-butyl, benzyl, or other suitable esters orethers. Short length of the compounds of the invention (2-7, preferably3 or 4 amino acids) is advantageous because it provides stability andincreased half life.

As mentioned earlier, the agents, including their individual targetingand reactive groups, may be synthesized using recombinant or chemicallibrary synthesis approaches. Libraries of interest in the inventioninclude peptide libraries, synthetic organic combinatorial libraries,and the like. The artisan or ordinary skill is familiar with themethodology for library and combinatorial chemistry synthesis as well asthe screening of such compounds for agents which are useful in themethods of the invention. The use of library technology, such as phagedisplay, and combinatorial chemistry, such as compound array methods, inthe synthesis and screening of protease inhibitors has been previouslydescribed in U.S. patent application entitled “Multivalent Compounds forCrosslinking Receptors and Uses Thereof” filed on Apr. 12, 1999 andassigned U.S. Ser. No. 09/290,376 (abandoned), the contents of which areincorporated in their entirety by reference. Examples of parallelsynthesis mixtures and parallel synthesis methods are provided in U.S.Ser. No. 08/177,497, filed Jan. 5, 1994 (abandoned) and itscorresponding PCT published patent application WO95/18972, publishedJul. 13, 1995 and U.S. Pat. No. 5,712,171 granted Jan. 27, 1998 and itscorresponding PCT published patent application WO96/22529, which arehereby incorporated by reference.

According to one aspect of the invention, a method for treating asubject having a condition characterized by an abnormal mammalian cellproliferation is provided. As used herein, subject means a mammalincluding humans, nonhuman primates, dogs, cats, sheep, goats, horses,cows, pigs and rodents. An abnormal mammalian cell proliferationdisorder or condition, as used herein, refers to a localized region ofcells (e.g., a tumor) which exhibit an abnormal (e.g., increased) rateof division as compared to their normal tissue counterparts.

Conditions characterized by an abnormal mammalian cell proliferation, asused herein, include but are not limited to conditions involving solidtumor masses of benign, pre-malignant or malignant character. Althoughnot wishing to be bound by a particular theory or mechanism, some ofthese solid tumor masses arise from at least one genetic mutation, somemay display an increased rate of cellular proliferation as compared tothe normal tissue counterpart, and still others may display factorindependent cellular proliferation. Factor independent cellularproliferation is an example of a manifestation of loss of growth controlsignals which some, if not all, tumors or cancers undergo.

Since the invention is premised, in part, on the finding that agents ofFormula I are able to inhibit FAP-α, a cell surface marker of reactivestromal fibroblasts, in one aspect, the invention involves treatingconditions involving a tumor mass which contains or is dependent uponthe presence of reactive stromal fibroblasts at some point during itsdevelopment. As used herein, reactive fibroblasts are fibroblasts whichhave been activated to express proteins such as receptors and growthfactors which, in some instances, have a positive effect and, in otherinstances, have a negative effect on cellular proliferation and growthof the fibroblasts themselves, and other cell types such as malignantcells of a carcinoma or epithelial metastasis.

In one aspect, the invention provides a method for treating subjectshaving a condition characterized by an abnormal epithelial cellproliferation. Epithelial cells are cells occurring in one or morelayers which cover the entire surface of the body and which line most ofthe hollow structures of the body, excluding the blood vessels, lymphvessels, and the heart interior which are lined with endothelium, andthe chest and abdominal cavities which are lined with mesothelium.Examples of epithelium include anterius corneae, anterior epithelium ofcornea, Barrett's epithelium, capsular epithelium, ciliated epithelium,columnar epithelium, epithelium corneae, corneal epithelium, cubicalepithelium, cubical epithelium, cuboidal epithelium, epithelium eductussemicircularis, enamel epithelium, false epithelium, germinalepithelium, gingival epithelium, glandular epithelium, glomerularepithelium, laminated epithelium, epithelium of lens, epithelium lentis,mesenchymal epithelium, olfactory epithelium, pavement epithelium,pigmentary epithelium, pigmented epithelium, protective epithelium,pseudostratified epithelium, pyramidal epithelium, respiratoryepithelium, rod epithelium, seminiferous epithelium, sense epithelium,sensory epithelium, simple epithelium, squamous epithelium, stratifiedepithelium, subcapsular epithelium, sulcular epithelium, tessellatedepithelium, transitional epithelium.

One category of conditions characterized by abnormal epithelial cellproliferation is proliferative dermatologic disorders. These includeconditions such as keloids, seborrheic keratosis, papilloma virusinfection (e.g. producing verruca vulbaris, verruca plantaris, verrucaplana, condylomata, etc.) and eczema.

An epithelial precancerous lesion is a skin lesion which has apropensity to develop into a cancerous condition. Epithelialprecancerous skin lesions also arise from other proliferative skindisorders such as hemangiomas, keloids, eczema and papilloma virusinfections producing verruca vulbaris, verruca plantaris and verrucaplanar. The symptoms of the epithelial precancerous lesions includeskin-colored or red-brown macule or papule with dry adherent scales.Actinic keratosis is the most common epithelial precancerous lesionamong fair skinned individuals. It is usually present as lesions on theskin which may or may not be visually detectable. The size and shape ofthe lesions varies. It is a photosensitive disorder and may beaggravated by exposure to sunlight. Bowenoid actinic keratosis isanother form of an epithelial precancerous lesion. In some cases, thelesions may develop into an invasive form of squamous cell carcinoma andmay pose a significant threat of metastasis. Other types of epithelialprecancerous lesions include hypertrophic actinic keratosis, arsenicalkeratosis, hydrocarbon keratosis, thermal keratosis, radiationkeratosis, viral keratosis, Bowen's disease, erythroplaquia of queyrat,oral erythroplaquia, leukoplakia, and intraepidermal epithelialoma.

Another category of conditions characterized by abnormal epithelial cellproliferation is tumors of epithelial origin. FAP-α has been observed intumors of epithelial origin Thus, in one aspect, the invention providesa method for treating subjects having epithelial tumors. Epithelialtumors are known to those of ordinary skill in the art and include, butare not limited to, benign and premalignant epithelial tumors, such asbreast fibroadenoma and colon adenoma, and malignant epithelial tumors.Malignant epithelial tumors include primary tumors, also referred to ascarcinomas, and secondary tumors, also referred to as metastases ofepithelial origin. Carcinomas intended for treatment with the methods ofthe invention include, but are not limited to, acinar carcinoma, acinouscarcinoma, alveolar adenocarcinoma (also called adenocystic carcinoma,adenomyoepithelioma, cribriform carcinoma and cylindroma), carcinomaadenomatosum, adenocarcinoma, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma (also called bronchiolar carcinoma,alveolar cell tumor and pulmonary adenomatosis), basal cell carcinoma,carcinoma basocellulare (also called basaloma, or basiloma, and hairmatrix carcinoma), basaloid carcinoma, basosquamous cell carcinoma,breast carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma,bronchogenic carcinoma, cerebriform carcinoma, cholangiocellularcarcinoma (also called cholangioma and cholangiocarcinoma), chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epibulbarcarcinoma, epidermoid carcinoma, carcinoma epitheliale adenoides,carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, gigantocellulare, glandularcarcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoidcarcinoma, hepatocellular carcinoma (also called hepatoma, malignanthepatoma and hepatocarcinoma), Hürthle cell carcinoma, hyalinecarcinoma, hypernephroid carcinoma, infantile embryonal carcinoma,carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma,Krompecher's carcinoma, Kulchitzky-cell carcinoma, lenticular carcinoma,carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma,carcinoma mastitoides, carcinoma medullare, medullary carcinoma,carcinoma melanodes, melanotic carcinoma, mucinous carcinoma, carcinomamuciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinomamucosum, mucous carcinoma, carcinoma myxomatodes, nasopharyngealcarcinoma, carcinoma nigrum, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, ovarian carcinoma, papillary carcinoma, periportalcarcinoma, preinvasive carcinoma, prostate carcinoma, renal cellcarcinoma of kidney (also called adenocarcinoma of kidney andhypernephroid carcinoma), reserve cell carcinoma, carcinomasarcomatodes, scheinderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, carcinoma vilosum. In preferredembodiments, the methods of the invention are used to treat subjectshaving cancer of the breast, cervix, ovary, prostate, lung, colon andrectum, pancreas, stomach or kidney.

Other conditions characterized by an abnormal mammalian cellproliferation to be treated by the methods of the invention includesarcomas. Sarcomas are rare mesenchymal neoplasms that arise in bone andsoft tissues. Different types of sarcomas are recognized and theseinclude: liposarcomas (including myxoid liposarcomas and pleiomorphicliposarcomas), leiomyosarcomas, rhabdomyosarcomas, malignant peripheralnerve sheath tumors (also called malignant schwannomas,neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors (includingEwing's sarcoma of bone, extraskeletal [not bone] Ewing's sarcoma, andprimitive neuroectodermal tumor [PNET]), synovial sarcoma,angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma,hemangioendothlioma, fibrosarcoma, desmoid tumor (also called aggressivefibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibroushistiocytoma (MFH), hemangiopericytoma, malignant mesenchymoma, alveolarsoft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplasticsmall cell tumor, gastrointestinal stromal tumor (GIST) (also known asGI stromal sarcoma), osteosarcoma (also known as osteogenicsarcoma)-skeletal and extraskeletal, and chondrosarcoma.

The methods of the invention are also directed towards the treatment ofsubjects with melanoma. Melanomas are tumors arising from themelanocytic system of the skin and other organs. Examples of melanomainclude lentigo maligna melanoma, superficial spreading melanoma,nodular melanoma, and acral lentiginous melanoma.

Other conditions characterized by an abnormal mammalian cellproliferation are cancers including, but not limited to, biliary tractcancer, endometrial cancer, esophageal cancer, gastric cancer,intraepithelial neoplasms, including Bowen's disease and Paget'sdisease, liver cancer, oral cancer, including squamous cell carcinoma,sarcomas, including fibrosarcoma and osteosarcoma, skin cancer,including melanoma, Kaposi's sarcoma, testicular cancer, includinggerminal tumors (seminoma, non-seminoma (teratomas, choriocarcinomas)),stromal tumors and germ cell tumors, thyroid cancer, including thyroidadenocarcinoma and medullar carcinoma, and renal cancer includingadenocarcinoma and Wilms tumor.

According to other aspects of the invention, a method is provided fortreating a subject having an abnormal proliferation originating in bone,muscle or connective tissue. Exemplary conditions intended for treatmentby the method of the invention include primary tumors (i.e., sarcomas)of bone and connective tissue.

The methods of the invention are also directed towards the treatment ofsubjects with metastatic tumors. In some embodiments, the metastatictumors are of epithelial origin. Carcinomas may metastasize to bone, ashas been observed with breast cancer, and liver, as is sometimes thecase with colon cancer. The methods of the invention are intended totreat metastatic tumors regardless of the site of the metastasis and/orthe site of the primary tumor. In preferred embodiments, the metastasesare of epithelial origin.

The method, in one embodiment, intends to treat subjects free ofsymptoms calling for hemopoietic stimulation, by administering compoundsof Formula I in an amount effective to inhibit proliferation. Theability to treat subjects having symptoms calling for hemopoieticstimulation with the some of compounds (e.g., ValboroPro) describedherein has been previously disclosed in U. S. patent applicationentitled “Hematopoietic Stimulation”, Ser. No. 09/304,199, filed May 3,1999, now issued as U.S. Pat. No. 6,300,314, on Oct. 9, 2001, thecontents of which are incorporated herein in their entirety byreference. Thus, the instant invention intends, in certain embodiments,to treat subjects at a time when they are free of symptoms requiringhemopoietic stimulating treatment or to treat subjects who have suchsymptoms with amounts or dosages or administration schedules that differfrom those used to protect or restore normal or protective levels ofhemopoietic cells. A subject who has previously experienced a need forhemopoietic stimulation but has since recovered its hemopoietic cells tonormal or at least protective levels may still be treated by the methodsdescribed herein.

As used herein, the terms hemopoietic and hematopoietic are usedinterchangeably to mean all blood cells including myeloid and lymphoidcells. Myeloid cells include erythrocytes (i.e., red blood cells),macrophages, monocytes, granulocytes including neutrophils, eosinophilsand basophils, mast cells, megakaryoctyes, platelets and dendriticcells, and lymphoid cells include T and B lymphocytes, thymic dendriticcells and natural killer (NK) cells. Hemopoietic stimulation, as usedherein, refers to the increase in hemopoietic cell numbers or activityto normal or protective levels.

An example of a symptom calling for hemopoietic stimulation ishemopoietic cell numbers below normal or protective levels. A “normal”level as used herein may be a level in a control population, whichpreferably includes subjects having similar characteristics as thetreated individual, such as age and sex. The “normal” level can also bea range, for example, where a population is used to obtain a baselinerange for a particular group into which the subject falls. Thus, the“normal” value can depend upon a particular population selected.Preferably, the normal levels are those of apparently healthy subjectswho have no prior history of hematopoietic cell disorders. Such “normal”levels, then can be established as preselected values, taking intoaccount the category in which an individual falls. Appropriate rangesand categories can be selected with no more than routine experimentationby those of ordinary skill in the art. Either the mean or anotherpreselected number within the range can be established as the normalpreselected value.

In general, the normal range for neutrophils is about 1800-7250 per μl(mean −3650); for basophils 0-150 per μl (mean −30); for eosinophils0-700 per μl (mean −150); for macrophages and monocytes 200-950 per μl(mean −430); for lymphocytes 1500-4000 per μl (mean −2500); forerythrocytes 4.2×10⁶−6.1×10⁶ per μl; and for platelets 133×10³−333×10³per μl. The foregoing ranges are at the 95% confidence level.

In connection with certain conditions, the medical community hasestablished certain preselected values. For example, mild neutropenia ischaracterized as having a count of between 1000 and 2000 per μl,moderate neutropenia at between 500 and 1000 per μl and severeneutropenia at below 500 per μl. Likewise, in adults, a lymphocyte countat less than 1500 is considered a medically undesirable condition. Inchildren, the value is less than 3000. Other preselected values will bereadily known to those of ordinary skill in the art.

A protective level of hematopoietic cells is the number of cellsrequired to confer clinical benefit to the patient. The required levelcan be equal to or less than the “normal level”. Such levels are wellknown to those of ordinary skill in the art. For example, a protectivelevel of neutrophils is above 1000, preferably, at least 1500.

Thus the methods of the invention, according to some embodiments, aredirected towards subjects who possess normal or protective levels ofhemopoietic cells, as described herein. Subjects with normal orprotective levels of hemopoietic cells are considered to have normalhemopoietic activity. Likewise, in some embodiments, the invention isdirected for use in subjects who are not immunocompromised. As usedherein, the terms immunocompromised and immunosuppressed are usedinterchangeably. An example of an immunocompromised subject is oneinfected with HIV and experiencing AIDS-related symptoms such as lowCD4+ T lymphocyte levels. In still other embodiments, the methods may beused in subjects who are HIV-positive and who may be immunocompromised,provided that the agent is administered in an amount, a dosing regimen,and an administration schedule that have a therapeutic effect onabnormal proliferation, such as in a Kaposi's sarcoma tumor, but are nottherapeutically effective in stimulating hemopoiesis in the subject.

According to still other embodiments, subjects of the invention arethose who may have previously received anti-cancer therapy or who willin the future receive anti-cancer therapy but who do not at the time oftreatment need hemopoietic stimulation, including a blood transfusion oradministration of a hemopoietic stimulant such as a hemopoietic growthfactor.

Thus in certain embodiments, the subjects are not myeloid or lymphoidsuppressed or are not candidates for treatment with an agent whichcauses such suppression at the time of treatment with the methods of theinstant invention. Myeloid suppressing conditions are those which inducea reduction in myeloid cells such as erythrocytes, neutrophils orplatelets, to below protective or normal levels. Exemplarymyelosuppressed conditions are hemopoietic malignancies, includingleukemia and lymphoma and diseases such as chronic idiopathicneutropenia, cyclic neutropenia, anemia and thrombocytopenia. Similarly,lymphoid suppressing conditions are those which induce a reduction inlymphoid cells such as T lymphocytes. Suppression of lymphoid cells orsome myeloid cells such as granulocytes is also referred to asimmunosuppression since reduction in these cell types makes anindividual susceptible to, inter alia, infection. Subjects may beexposed to myeloid, lymphoid or general immune suppressing conditions bythe use of either immunosuppressant drugs such as cyclosporin or highdose chemotherapeutic compounds which affect dividing hemopoietic cells.Immuno-suppression may also arise as a result of treatment modalitiessuch as total body irradiation or conditioning regimens prior to bonemarrow transplantation. Viral infection, particularly as in the case ofinfection with human immunodeficiency virus (HIV), may alsoimmunosuppress an individual. In some embodiments, subjects are thosewhich have not been exposed and are not anticipated to be exposed to theabove-mentioned conditions. In other embodiments, the instant inventionaims to treat subjects who may have been myelosuppressed orimmunosuppressed (e.g., by exposure to one or more of the aboveconditions), provided that at the time of treatment using the methodsdescribed herein, the subject has protective or normal levels ofhemopoietic cells.

In still other embodiments, the invention aims to treat subjects who mayexhibit symptoms calling for hemopoietic stimulation, provided that theagents are administered in doses, routes and schedules that would notresult in hemopoietic stimulation, as explained below. In certainembodiments, the methods of the invention are not intended for use inthe treatment of malignancies in HIV infected (i.e., HIV positive orHIV+) subjects who have below normal or below protective levels ofhemopoietic cells, unless the agents are used under conditions, such asadministration routes, doses or dosing schedules, that aretherapeutically effective in treating abnormal cell proliferation, asdescribed herein, and not effective in stimulating hemopoiesis. Forexample, in some embodiments, the agent may be administered once a day,or twice a day, or three or more times a day, for more than 7 days, morethan 10 days, more than 14 days or more than 20 days in order toachieve, for example, sustained desired systemic levels. In otherembodiments, the agent may be given at timed intervals, such as, forexample, every two days, every three days, every four days, every weekor every two weeks. In still further embodiments, the agent may bedelivered intravenously and continuously, for example, or by injection,such as, in single bolus administrations.

According to another aspect of the invention, methods are provided forinhibiting angiogenesis in disorders having a pathology which requiresangiogenesis. Angiogenesis is defined as the formation of new bloodvessels. One subset of these disorders is conditions characterized byabnormal mammalian cell proliferation. Another subset is non-cancerconditions including diabetic retinopathy, neovascular glaucoma andpsoriasis.

In preferred embodiments, the methods of the invention are aimed atinhibiting tumor angiogenesis. Tumor angiogenesis refers to theformation of new blood vessels in the vicinity or within a tumor mass.Solid tumor cancers require angiogenesis particularly for oxygen andnutrient supply. It has been previously shown that inhibition ofangiogenesis in solid tumor can cause tumor regression in animal models.Thus in one aspect, the invention relates to a method for inhibitingangiogenesis by inhibiting the proliferation, migration or activation ofendothelial cells and fibroblasts, provided this angiogenesis isunrelated to wound healing in response to injury, infection orinflammation.

Thus in certain embodiments, the methods of the invention are intendedfor the treatment of diseases and processes that are mediated byangiogenesis including, but not limited to, hemangioma, solid tumors,tumor metastasis, benign tumors, for example hemangiomas, acousticneuromas, neurofibromas and trachomas, Osler-Webber Syndrome,telangiectasia, myocardial angiogenesis, angiofibroma, plaqueneovascularization, coronary collaterals, ischemic limb angiogenesis,corneal diseases, rubiosis, neovascular glaucoma, diabetic retinopathy,retrolental fibroplasia, diabetic neovascularization, maculardegeneration, keloids, ovulation, menstruation, and placentation.

The compositions and methods of the invention in certain instances maybe useful for replacing existing surgical procedures or drug therapies,although in most instances the present invention is useful in improvingthe efficacy of existing therapies for treating such conditions.Accordingly combination therapy may be used to treat the subjects. Forexample, the agent may be administered to a subject in combination withanother anti-proliferative (e.g., an anti-cancer) therapy. Suitableanti-cancer therapies include surgical procedures to remove the tumormass, chemotherapy or localization radiation. The otheranti-proliferative therapy may be administered before, concurrent with,or after treatment with the agent of the invention. There may also be adelay of several hours, days and in some instances weeks between theadministration of the different treatments, such that the agent may beadministered before or after the other treatment.

As an example, the agent may be administered in combination with surgeryto remove an abnormal proliferative cell mass. As used herein, “incombination with surgery” means that the agent may be administered priorto, during or after the surgical procedure. Surgical methods fortreating epithelial tumor conditions include intra-abdominal surgeriessuch as right or left hemicolectomy, sigmoid, subtotal or totalcolectomy and gastrectomy, radical or partial mastectomy, prostatectomyand hysterectomy. In these embodiments, the agent may be administeredeither by continuous infusion or in a single bolus. Administrationduring or immediately after surgery may include a lavage, soak orperfusion of the tumor excision site with a pharmaceutical preparationof the agent in a pharmaceutically acceptable carrier. In someembodiments, the agent is administered at the time of surgery as well asfollowing surgery in order to inhibit the formation and development ofmetastatic lesions. The administration of the agent may continue forseveral hours, several days, several weeks, or in some instances,several months following a surgical procedure to remove a tumor mass.

The subjects can also be administered the agent in combination withnon-surgical anti-proliferative (e.g., anti-cancer) drug therapy. In oneembodiment, the agent may be administered in combination with ananti-cancer compound such as a cytostatic compound. A cytostaticcompound is a compound (e.g., a nucleic acid, a protein) that suppressescell growth and/or proliferation. In some embodiments, the cytostaticcompound is directed towards the malignant cells of a tumor. In yetother embodiments, the cytostatic compound is one which inhibits thegrowth and/or proliferation of vascular smooth muscle cells orfibroblasts.

Suitable anti-proliferative drugs or cytostatic compounds to be used incombination with the agents of the invention include anti-cancer drugs.Anti-cancer drugs are well known and include; Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine;Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine;Anastrozole; Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa;Azotomycin; Batimastat; Benzodepa; Bicalutamide; BisantreneHydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate;Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Taxol; Taxotere; Tecogalan Sodium; Tegafur;Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone;Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin;Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; TrestoloneAcetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate;Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa;Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate;Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride.

Other anti-cancer drugs include: 20-epi-1,25 dihydroxyvitamin D3;5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine;amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine;anagrelide; anastrozole; andrographolide; angiogenesis inhibitors;antagonist D; antagonist G; antarelix; anti-dorsalizing morphogeneticprotein-1; antiandrogen, prostatic carcinoma; antiestrogen;antineoplaston; antisense oligonucleotides; aphidicolin glycinate;apoptosis gene modulators; apoptosis regulators; apurinic acid;ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane;atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron;azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat;BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactamderivatives; beta-alethine; betaclamycin B; betulinic acid; bFGFinhibitor; bicalutamide; bisantrene; bisaziridinyispermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor;carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropinB; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; cladribine; clomifene analogues; clotrimazole;collismycin A; collismycin B; combretastatin A4; combretastatinanalogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8;cryptophycin A derivatives; curacin A; cyclopentanthraquinones;cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin;diphenyl spiromustine; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin;epristeride; estramustine analogue; estrogen agonists; estrogenantagonists; etanidazole; etoposide phosphate; exemestane; fadrozole;fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;flezelastine; fluasterone; fludarabine; fluorodaunorunicinhydrochloride; forfenimex; formestane; fostriecin; fotemustine;gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam;heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid;idarubicin; idoxifene; idramantone; ilmofosine; ilomastat;imidazoacridones; imiquimod; immunostimulant peptides; insulin-likegrowth factor-1 receptor inhibitor; interferon agonists; interferons;interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan;iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron;jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;leukemia inhibiting factor; leukocyte alpha interferon;leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole;linear polyamine analogue; lipophilic disaccharide peptide; lipophilicplatinum compounds; lissoclinamide 7; lobaplatin; lombricine;lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine;lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides;maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysininhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone;miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone;mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growthfactor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonalantibody, human chorionic gonadotrophin; monophosphoryl lipidA+myobacterium cell wall sk; mopidamol; multiple drug resistance geneinhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer compound; mycaperoxide B; mycobacterial cell wall extract;myriaporone; N-acetyldinaline; N-substituited benzamides; nafarelin;nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim;nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant;nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides;onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer;ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues;paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid;panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase;peldesine; pentosan polysulfate sodium; pentostatin; pentrozole;perflubron; perfosfamide; perillyl alcohol; phenazinomycin;phenylacetate; phosphatase inhibitors; picibanil; pilocarpinehydrochloride; pirarubicin; piritrexim; placetin A; placetin B;plasminogen activator inhibitor; platinum complex; platinum compounds;platinum-triamine complex; porfimer sodium; porfiromycin; propylbis-acridone; prostaglandin J2; proteasome inhibitors; protein A-basedimmune modulator; protein kinase C inhibitor; protein kinase Cinhibitors, microalgal; protein tyrosine phosphatase inhibitors; purinenucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine;pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists;raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors;ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide;rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol;saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics;semustine; senescence derived inhibitor 1; sense oligonucleotides;signal transduction inhibitors; signal transduction modulators; singlechain antigen binding protein; sizofiran; sobuzoxane; sodiumborocaptate; sodium phenylacetate; solverol; somatomedin bindingprotein; sonermin; sparfosic acid; spicamycin D; spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-celldivision inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;superactive vasoactive intestinal peptide antagonist; suradista;suramin; swainsonine; synthetic glycosaminoglycanis; tallimustine;tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietinmimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;titanocene dichloride; topotecan; topsentin; toremifene; totipotent stemcell factor; translation inhibitors; tretinoin; triacetyluridine;triciribine; trimetrexate; triptorelin; tropisetron; turosteride;tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;urogenital sinus-derived growth inhibitory factor; urokinase receptorantagonists; vapreotide; variolin B; vector system, erythrocyte genetherapy; velaresol; veramine; verdins; verteporfin; vinorelbine;viaxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;zinostatin stimalamer.

Anti-cancer supplementary potentiating compounds include: Tricyclicanti-depressant drugs (e.g., imipramine, desipramine, amitryptyline,clomipramine, trimipramine, doxepin, nortriptyline, protriptyline,amoxapine and maprotiline); non-tricyclic anti-depressant drugs (e.g.,sertraline, trazodone and citalopram); Ca++ antagonists (e.g.,verapamril, nifedipine, nitrendipine and caroverine); Calmodulininhibitors (e.g., prenylamine, trifluoroperazine and clomipramine);Amphotericin B; Triparanol analogues (e.g., tamoxifen); antiarrhythmicdrugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thioldepleters (e.g., buthionine and sulfoximine) and multiple drugresistance reducing compounds such as Cremaphor EL.

Other compounds which are useful in combination therapy for the purposeof the invention include the antiproliferation compound, PiritreximIsethionate; the antiprostatic hypertrophy compound, Sitogluside; thebenign prostatic hyperplasia therapy compound, Tamsulosin Hydrochloride;the prostate growth inhibitor, Pentomone; radioactive compounds such asFibrinogen 1 125, Fludeoxyglucose F 18, Fluorodopa F 18, Insulin I 125,Insulin I 131, Iobenguane I 123, Iodipamide Sodium I 131, IodoantipyrineI 131, Iodocholesterol I 131, Iodohippurate Sodium I 123, IodohippurateSodium I 125, Iodohippurate Sodium I 131, Iodopyracet I 125, IodopyracetI 131, Iofetamine Hydrochloride I 123, Iomethin I 125, Iomethin I 131,Iothalamate Sodium I 125, Iothalamate Sodium I 131, Iotyrosine I 131,Liothyronine I 125, Liothyronine I 131, Merisoprol Acetate Hg 197,Merisoprol Acetate Hg 203, Merisoprol Hg 197, Selenomethionine Se 75,Technetium Tc 99 m Antimony Trisulfide Colloid, Technetium Tc 99 mBicisate, Technetium Tc 99 m Disofenin, Technetium Tc 99 m Etidronate,Technetium Tc 99 m Exametazime, Technetium Tc 99 m Furifosmin,Technetium Tc 99 m Gluceptate, Technetium Tc 99 m Lidofenin, TechnetiumTc 99 m Mebrofenin, Technetium Tc 99 m Medronate, Technetium Tc 99 mMedronate Disodium, Technetium Tc 99 m Mertiatide, Technetium Tc 99 mOxidronate, Technetium Tc 99 m Pentetate, Technetium Tc 99 m PentetateCalcium Trisodium, Technetium Tc 99 m Sestamibi, Technetium Tc 99 mSiboroxime, Technetium Tc 99 m Succimer, Technetium Tc 99 m SulfurColloid, Technetium Tc 99 m Teboroxime, Technetium Tc 99 m Tetrofosmin,Technetium Tc 99 m Tiatide, Thyroxine I 125, Thyroxine I 131,Tolpovidone I 131, Triolein I 125 and Triolein I 131.

According to the methods of the invention, the agents of Formula I maybe administered prior to, concurrent with, or following the otheranti-cancer compounds. The administration schedule may involveadministering the different agents in an alternating fashion. In otherembodiments, the agent may be delivered before and during, or during andafter, or before and after treatment with other therapies. In somecases, the agent is administered more than 24 hours before theadministration of the other anti-proliferative treatment. In otherembodiments, more than one anti-proliferative therapy may beadministered to a subject. For example, the subject may receive theagents of the invention, in combination with both surgery and at leastone other anti-proliferative compound. Alternatively, the agent may beadministered in combination with more than one anti-cancer drug.

Other compounds useful in combination therapies with the inhibitorcompounds of the invention include anti-angiogenic compounds such asangiostatin, endostatin, fumagillin, non-glucocorticoid steroids andheparin or heparin fragments and antibodies to one or more angiogenicpeptides such as αFGF, βFGF, VEGF, IL-8 and GM-CSF. These latteranti-angiogenic compounds may be administered along with the inhibitoragents of the invention (i.e., the agents of Formula I) for the purposeof inhibiting proliferation or inhibiting angiogenesis in all of theaforementioned conditions as described herein. In certain embodiments,the agent may be administered in combination with an anti-angiogeniccompound and at least one of the anti-proliferative therapies describedabove including surgery or anti-proliferative drug therapy.

The above-described drug therapies are well known to those of ordinaryskill in the art and are administered by modes known to those of skillin the art. The drug therapies are administered in amounts which areeffective to achieve physiological goals such as the inhibition ofproliferation or inhibition of angiogenesis, in combination with theagents of the invention. It is contemplated that the drug therapies maybe administered in amounts which, when used alone, may not be capable ofinhibiting proliferation or angiogenesis but which, when administered incombination with the agents of the invention, are capable of achievingthe desired level of inhibition. Thus, in embodiments in which the agentof Formula I is administered with another therapeutic agent (e.g., ananti-proliferative compound or an anti-angiogenic compound),subtherapeutic doses of either or both agents may be used. In stillother embodiments, anti-proliferative drug therapies may be administeredin conditions such as doses or amounts which do not affect hemopoieticcell levels in the subjects.

The agents of the invention are administered in therapeuticallyeffective amounts. An effective amount is a dosage of the agentsufficient to provide a medically desirable result. The effective amountwill vary with the particular condition being treated, the age andphysical condition of the subject being treated, the severity of thecondition, the duration of the treatment, the nature of the concurrentor combination therapy (if any), the specific route of administrationand like factors within the knowledge and expertise of the healthpractitioner. It is preferred generally that a maximum dose be used,that is, the highest safe dose according to sound medical judgment.

For example, in connection with methods directed towards treatingsubjects having a condition characterized by abnormal mammalian cellproliferation, an effective amount to inhibit proliferation would be anamount sufficient to reduce or halt altogether the abnormal mammaliancell proliferation so as to slow or halt the development of or theprogression of a cell mass such as, for example, a tumor. As used in theembodiments, “inhibit” embraces all of the foregoing.

According to other aspects of the invention directed at inhibitingangiogenesis in a subject having a condition characterized by anabnormal mammalian cell proliferation, an effective amount to inhibitangiogenesis would be an amount sufficient to lessen or inhibitaltogether smooth muscle cell proliferation so as to slow or halt thedevelopment of or the progression of tumor vascularization. As used inthese embodiments, “inhibit” embraces all of the foregoing.

When used therapeutically, the agent is administered in therapeuticallyeffective amounts. In general, a therapeutically effective amount meansthat amount necessary to delay the onset of, inhibit the progression of,or halt altogether the particular condition being treated. In someaspects of the invention, an therapeutically effective amount will bethat amount necessary to inhibit mammalian cell proliferation. In otherembodiments, a therapeutically effective amount will be an amountnecessary to extend the dormancy of micrometastases or to stabilize anyresidual primary tumor cells following surgical or drug therapy.

In still other embodiments, the agent is delivered in an amount, a dose,and a schedule which is therapeutically effective in inhibitingproliferation yet which is not therapeutically effective in stimulatinghemopoiesis in the subject. In administering the agents of the inventionto subjects, dosing amounts, dosing schedules, routes of administrationand the like can be selected so as to affect the other known activitiesof these compounds. For example, amounts, dosing schedules and routes ofadministration can be selected as described below, wherebytherapeutically effective levels for inhibiting proliferation areprovided, yet therapeutically effective levels for restoring hemopoieticdeficiency are not provided. As another example, local administration totumors or protected body areas such as the brain may result intherapeutically effective levels for inhibiting proliferation, but maybe non-therapeutically effective levels for hemopoietic cellstimulation.

In addition, agents of Formula I can be selected that are effective asanti-proliferative agents but are relatively ineffective as hemopoieticcell stimulatory or activating agents. Thus, certain subjects whorequire both hemopoietic stimulation and/or activation and proliferationinhibition may be treated with different agents of Formula Isimultaneously, one each for the desired therapeutic effect, or with asingle agent but in different dosages, schedules, and/or route toachieve both hemopoietic stimulation and proliferation inhibition attherapeutic levels.

Generally, a therapeutically effective amount will vary with thesubject's age, condition, and sex, as well as the nature and extent ofthe disease in the subject, all of which can be determined by one ofordinary skill in the art. The dosage may be adjusted by the individualphysician or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount typically varies from0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg,in one or more dose administrations daily, for one or more days. In someembodiments, the agents are administered for more than 7 days, more than10 days, more than 14 days and more than 20 days. In still otherembodiments, the agent is administered over a period of weeks, ormonths. In still other embodiments, the agent is delivered on alternatedays. For example, the agent is delivered every two days, or every threedays, or every four days, or every five days, or every six days, orevery week, or every month.

The agents of the invention can also be administered in prophylacticallyeffective amounts, particularly in subjects diagnosed with benign orpre-malignant tumors. In these instances, the agents are administered inan amount effective to prevent the development of an abnormal mammaliancell proliferative mass or to prevent angiogenesis in the solid tumormass, depending on the embodiment. The agents may also be administeredin an amount effective to prevent metastasis of cells from a tumor toother tissues in the body. In these latter embodiments, the invention isdirected to preventing the metastatic spread of a primary tumor.

According to another aspect of the invention, a kit is provided. The kitis a package which houses a container which contains an agent of theinvention and also houses instructions for administering the agent ofthe invention to a subject having a condition characterized by anabnormal mammalian cell proliferation. The kit may optionally alsocontain one or more other anti-proliferative compounds or one or moreanti-angiogenic compounds for use in combination therapies as describedherein.

In still another aspect of the invention, kits for administration of anagent of the invention to a subject is provided. The kits include acontainer containing a composition which includes at least one agent ofthe invention, and instructions for administering the at least one agentto a subject having a condition characterized by an abnormal mammaliancell proliferation in an amount effective to inhibit proliferation. Incertain embodiments, the container is a container for intravenousadministration. In other embodiments the agent is provided in aninhaler. In still other embodiments, the agent is provided in apolymeric matrix or in the form of a liposome. In yet other embodiments,kits are provided for the administration of an agent of the invention toa subject having an abnormal mammalian cell mass for the purpose ofinhibiting angiogenesis in the cell mass. In these latter kits, theagent is provided in an amount effective to inhibit angiogenesis alongwith instructions for use in subjects in need of such treatment.

The agent may be administered alone or in combination with theabove-described drug therapies by a variety of administration routesavailable. The particular mode selected will depend, of course, upon theagent selected, the condition being treated, the severity of thecondition, whether the treatment is therapeutic or prophylactic, and thedosage required for efficacy. The methods of the invention, generallyspeaking, may be practiced using any mode of administration that ismedically acceptable, meaning any mode that produces effective levels ofthe active compounds without causing clinically unacceptable adverseeffects. The administration may, for example, be oral, intraperitoneal,intra-cavity such as rectal or vaginal, transdermal, topical, nasal,inhalation, mucosal, interdermal, or parenteral routes. The term“parenteral” includes subcutaneous, intravenous, intramuscular, orinfusion. Intravenous or intramuscular routes may not particularlysuitable for long term therapy and prophylaxis. In certain embodiments,however, it may be appropriate to administer the agent in a continuousinfusion every several days, or once a week, or every several weeks, oronce a month. Intravenous or intramuscular routes may be preferred inemergency situations. Oral administration may be used for prophylactictreatment because of the convenience to the patient as well as thedosing schedule. Likewise, sustained release devices as described hereinmay be useful in certain embodiments for prophylactic or post surgerytreatment, for example.

When using the agent of the invention in subjects in whom the primarysite of abnormal proliferation is well delineated and easily accessible,direct administration to the site may be preferred, provided the tumorhas not already metastasized. For example, administration by inhalationfor lung tumors or by suppositories in the treatment of cervical,ovarian or rectal tumors may be preferred. Likewise, melanoma, forexample, may be treated with the agent via topical administration in andaround the area of the lesion. In still other embodiments aimed at thetreatment of subjects with breast or prostate cancer, the agents may bedelivered by injection directly into the tissue with, for example, abiopsy needle and syringe.

Systemic administration may be preferred in some instances such as, forexample, if the subject is known to have or is suspected of havingmetastases. In this way, all tumor sites, whether primary or secondarymay receive the agent. Systemic delivery may be accomplished through forexample, oral or parenteral administration. Inhalation may be used ineither systemic or local delivery, as described below.

As discussed earlier, the agent may also be delivered to a tumor siteduring or immediately after a surgical procedure to remove the tumor bylavage into the excision site or by perfusion of the affected tissuewith a physiologically acceptable solution containing the agent.Alternatively, the patient may be administered the agent prior to orfollowing the surgical procedure by continuous infusion. In yet otherembodiments, a sustained release device, as described below, such as apolymeric implant may be positioned during surgery in the vicinity ofthe excision site so as to provide a high local concentration of theagent. These latter embodiments may be appropriate to prevent regrowthof the tumor.

The agent of the invention may be administered alone or in combinationwith the above-described drug therapies as part of a pharmaceuticalcomposition. Such a pharmaceutical composition may include the agent incombination with any standard physiologically and/or pharmaceuticallyacceptable carriers which are known in the art. The compositions shouldbe sterile and contain either a therapeutically or prophylacticallyeffective amount of the agent in a unit of weight or volume suitable foradministration to a subject. The term “pharmaceutically-acceptablecarrier” as used herein means one or more compatible solid or liquidfiller, diluents or encapsulating substances which are suitable foradministration into a subject of the invention. The term “carrier”denotes an organic or inorganic ingredient, natural or synthetic, withwhich the active ingredient is combined to facilitate the application.The components of the pharmaceutical compositions also are capable ofbeing co-mingled with the molecules of the present invention, and witheach other, in a manner such that there is no interaction which wouldsubstantially impair the desired pharmaceutical efficacy.Pharmaceutically-acceptable further means a non-toxic material that iscompatible with a biological system such as a cell, cell culture,tissue, or organism. The characteristics of the carrier will depend onthe route of administration. Physiologically andpharmaceutically-acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the agent, which is preferablyisotonic with the blood of the recipient. This aqueous preparation maybe formulated according to known methods using suitable dispersing orwetting compounds and suspending compounds. The sterile injectablepreparation also may be a sterile injectable solution or suspension in anon-toxic parenterally-acceptable diluent or solvent, for example, as asolution in 1,3-butane diol. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution, and isotonic sodiumchloride solution. In addition, sterile, fixed oils are conventionallyemployed as a solvent or suspending medium. For this purpose, any blandfixed oil may be employed including synthetic mono- or di-glycerides. Inaddition, fatty acids such as oleic acid may be used in the preparationof injectables. Carrier formulations suitable for oral, subcutaneous,intravenous, intramuscular, etc. administrations can be found inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating compounds, andinert gases and the like. The pharmaceutical compositions mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well-known in the art of pharmacy.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the agent. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

In yet other embodiments, the preferred vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT InternationalApplication No. PCT/US/03307 (Publication No. WO 95/24929, entitled“Polymeric Gene Delivery System”, claiming priority to U.S. patentapplication Ser. No. 213,668, filed Mar. 15, 1994). PCT/US/0307describes a biocompatible, preferably biodegradable polymeric matrix forcontaining a biological macromolecule. The polymeric matrix may be usedto achieve sustained release of the agent in a subject. In accordancewith one aspect of the instant invention, the agent described herein maybe encapsulated or dispersed within the biocompatible, preferablybiodegradable polymeric matrix disclosed in PCT/US/03307. The polymericmatrix preferably is in the form of a microparticle such as amicrosphere (wherein the agent is dispersed throughout a solid polymericmatrix) or a microcapsule (wherein the agent is stored in the core of apolymeric shell). Other forms of the polymeric matrix for containing theagent include films, coatings, gels, implants, and stents. The size andcomposition of the polymeric matrix device is selected to result infavorable release kinetics in the tissue into which the matrix device isimplanted. The size of the polymeric matrix devise further is selectedaccording to the method of delivery which is to be used, typicallyinjection into a tissue or administration of a suspension by aerosolinto the nasal and/or pulmonary areas. The polymeric matrix compositioncan be selected to have both favorable degradation rates and also to beformed of a material which is bioadhesive, to further increase theeffectiveness of transfer when the devise is administered to a vascularor pulmonary surface. The matrix composition also can be selected not todegrade, but rather, to release by diffusion over an extended period oftime.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the agents of the invention to the subject. Biodegradablematrices are preferred. Such polymers may be natural or syntheticpolymers. Synthetic polymers are preferred. The polymer is selectedbased on the period of time over which release is desired, generally inthe order of a few hours to a year or longer. Typically, release over aperiod ranging from between a few hours and three to twelve months ismost desirable. The polymer optionally is in the form of a hydrogel thatcan absorb up to about 90% of its weight in water and further,optionally is cross-linked with multi-valent ions or other polymers.

In general, the agents of the invention may be delivered using thebioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitro celluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, poly vinylchloride, polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate). Thus, the invention provides acomposition of the above-described agents of Formula I for use as amedicament, methods for preparing the medicament and methods for thesustained release of the medicament in vivo.

Other delivery systems can include timed release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the agent of the invention, increasing convenience tothe subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude the above-described polymeric systems, as well as polymer basesystems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono- di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the agent is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whichan active component permeates at a controlled rate from a polymer suchas described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. Inaddition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for treatment of chronic conditions, such as a the suspectedpresence of dormant metastases. Long-term release, are used herein,means that the implant is constructed and arranged to deliverytherapeutic levels of the active ingredient for at least 30 days, atleast 60 days and more preferably for several months. Long-termsustained release implants are well-known to those of ordinary skill inthe art and include some of the release systems described above.

In still other embodiments, the agent is targeted to a site of abnormalcell proliferation, such as, a tumor, through the use of a targetingcompound specific for a particular tissue or tumor type. The agents ofthe invention may be targeted to primary or in some instances, secondary(i.e., metastatic) lesions through the use of targeting compounds whichpreferentially recognize a cell surface marker. The targeting compoundmay be directly conjugated to the agents of the invention via a covalentlinkage. The agent may be indirectly conjugated to a targeting compoundvia a linker. Alternatively, the targeting compound may be conjugated orassociated with an intermediary compound such as, for example, aliposome within which the agent is encapsulated. Liposomes areartificial membrane vessels which are useful as a delivery vector invivo or in vitro. It has been shown that large unilamellar vessels(LUV), which range in size from 0.2-4.0 μm can encapsulate largemacromolecules. Liposomes may be targeted to a particular tissue, suchas the vascular cell wall, by coupling the liposome to a specific ligandsuch as a monoclonal antibody, sugar, glycolipid, or protein. Liposomesare commercially available from Gibco BRL, for example, as LIPOFECTIN™and LIPOFECTACE™, which are formed of cationic lipids such as N-[1-(2,3dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride (DOTMA) anddimethyl dioctadecylammonium bromide (DDAB). Methods for makingliposomes are well known in the art and have been described in manypublications. Liposomes also have been reviewed by Gregoriadis, G. inTrends in Biotechnology, V. 3, p. 235-241 (1985). In still otherembodiments, the targeting compound may be loosely associated with theagents of the invention, such as within a microparticle comprising apolymer, the agent of the invention and the targeting compound.

Targeting compounds useful according to the methods of the invention arethose which direct the agent to a site of abnormal proliferation such asa tumor site. The targeting compound of choice will depend upon thenature of the tumor or the tissue origin of the metastasis. In someinstances it may be desirable to target the agent to the tissue in whichthe tumor is located. For example, agents can be delivered to breastepithelium by using a targeting compound specific for breast tissue. Inpreferred embodiments, the target is specific for malignant breastepithelium. Examples of compounds which may localize to malignant breastepithelium include, but are not limited to, estrogen and progesterone,epithelial growth factor (EGF) and HER-2/neu ligand, among others. TheHER-2/neu ligand may also be used to target agents to ovarian cancers.Ovarian cancers are also known to express EGFR and c-fms, and thus couldbe targeted through the use of ligands for either receptor. In the caseof c-fms which is also expressed by macrophages and monocytes, targeteddelivery to an ovarian cancer may require a combination of localadministration such as a vaginal suppository as well as a targetingcompound. Prostate cancers can be targeted using compounds such aspeptides (e.g., antibodies or antibody fragments) which bind to prostatespecific antigen (PSA) or prostate specific membrane antigen (PSMA).Other markers which may be used for targeting of the agent to specifictissues include, for example, in liver: HGF, insulin-like growth factorI, II, insulin, OV-6, HEA-125, hyaluronic acid, collagen, N-terminalpropeptide of collagen type III, mannose/N-acetylglucosamine,asialoglycoprotein, tissue plasminogen activator, low densitylipoprotein, carcinoembryonic antigen; in kidney cells: angiotensin II,vasopressin, antibodies to CD44v6; in keratinocytes and skinfibroblasts: KGF, very low density lipoprotein, RGD-containing peptides,collagen, laminin; in melanocytes: kit ligand; in gut:cobalamin-intrinsic factor, heat stable enterotoxin of E. Coli; inbreast epithelium: heregulin, prolactin, transferrin, cadherin-11. Othermarkers specific to particular tissues are available and would be knownto one of ordinary skill in the art.

In still other embodiments, the agent of the invention may be targetedto fibroblasts specifically, via ligands or binding partners forfibroblast specific markers. Examples of these markers include, but arenot limited to fibroblast growth factors (FGF) and platelet derivedgrowth factor (PDGF). In some embodiments, it may be desirable to targetthe agent to FAP-α specifically through the use of binding peptides forFAP-α which do not interfere with inhibition by the agent of theinvention. One such binding peptide is the monoclonal antibody F19, usedpreviously for immunodetection of reactive stromal fibroblast expressingFAP-α.

Agents useful in the invention can be identified using a screening assaymethod for determining whether a putative agent is able to inhibit theactivity of FAP-α, thereby inhibiting cell proliferation. The initialscreening assay can be conducted in an in vitro system with a readout ofFAP-α inhibition. In such screening assays, cells expressing FAP-α butnot CD26 can be used as a source of FAP-α. Alternatively, recombinant orpurified FAP-α can also be used in either a soluble or bound form. Thechoice of whether to use FAP-α in either a soluble or bound form maydepend upon the source of the compounds to be screened. For example, ifthe compounds to be screened are present in phage libraries, it may bedesirable to use soluble FAP-α. If, on the other hand, the compounds aresynthesized by combinatorial chemistry techniques, then bound FAP-α maybe more suitable. It is possible to immobilize FAP-α in 96 well platesthrough either direct binding to the surface, or more preferably throughthe indirect binding via an anti-FAP-α antibody or antibody fragmentsuch as that derived from F19, a FAP-α specific antibody. Binding isachieved through incubation at room temperature for 2 hours, followed bywashing with a phosphate buffered saline solution containing a suitablenon-specific blocking agent such as albumin or serum. After significantwashing, the substrate alanylprolyl-7-amido-4-trifluoromethyl-coumarin(Ala-Pro-NH-F3-Mec, available from Bachem) is added to the plates andincubated for 1 hour at 37° C. in 100 mM Tris/HCl, pH7.8, 100 mM NaCl.At the end of the incubation, a fluorometric measurement is made foreach well using an excitation wavelength of 390 nm and an emissionwavelength of 538 nm. The substrate described above can also be used insoluble FAP-α enzyme inhibition assays are described in U.S. patentapplication entitled “Multivalent Compounds for Crosslinking Receptorsand Uses Thereof” filed on Apr. 12, 1999 and assigned U.S. Ser. No.09/290,376 (abandoned).

The above described procedure represents the control for the inhibitionscreen. To perform the inhibition screen, the agents of Formula I areincubated with FAP-α for 5-10 minutes at 37° C. prior to introduction ofthe substrate. The enzyme reaction proceeds as above, and a fluorescentmeasurement is used as a readout. A decrease in the amount offluorescence is indicative of an inhibitor. Alternatively, the readoutcan be a kinetic analysis of the rate of fluorescence change, with aslower rate being indicative of an inhibitory agent.

Once FAP-α inhibitors have been pre-screened in vitro, they can betested in proliferation assays in vitro or in vivo. In vitroproliferation assays could analyze the effect of Formula I compounds onthe rate of proliferation of FAP-α expressing cells. Proliferation inthese assays can be measured either by tritiated thymidine uptake orsimply by a cell count. Carcinoma cell lines are not suitable for thesetypes of in vitro assays since these cells are generally FAP-α negative.Alternatively, inhibition of FAP-α protease activity in vitro or in vivoor inhibition of angiogenesis in vitro could also be used. Assay systemsfor angiogenesis inhibition are known in the art and are described inU.S. Pat. No. 5,854,221 and 5,639,725, the entire contents of which areincorporated herein by reference. In vivo assay systems involve theinitial induction of a suitable experimental tumor within a mouse,usually by the injection of a malignant cell line into a pre-definedlocation such as the lungs or the footpad. Following the implantationand growth of the tumor, the agent to be tested is administered to themouse, again usually over a period of time, and at differing doses. Atthe end of the assay, the mouse is analyzed in terms of, among otherthings, tumor growth and the presence of metastases. In assay systemsaimed at studying the prophylactic efficacy of an agent, the agent maybe administered in close temporal proximity to the tumor cell lineinjection. In this way, one can determine whether the agent is able toprevent tumor formation altogether. These assay systems are described inmore detail in the following Examples.

Identifying compounds and administration regimens which favorproliferation inhibition over hemopoietic stimulation, including dosingamounts, dosing schedules and routes of administration, involvescomparison of results of the above assays to hemopoietic stimulationassays described previously in U.S. patent application Ser. No.09/304,199, filed May 3, 1999, entitled “Hematopoietic Stimulation”, nowissued as U.S. Pat. No. 6,300,314, on Oct. 9, 2001, the contents ofwhich are incorporated herein in their entirety by reference.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

All patents, patent applications, references and other documentsidentified herein are incorporated in their entirety herein byreference.

EXAMPLES Example 1

This example illustrates the use of PT-100 (i.e., ValboroPro) to inhibitFAP-α protease activity and stromal cytokine secretion from FAP-αpositive CD26/DPPIV negative rat and human stromal cells.

Materials and Methods: Rat Stromal Cultures

Cell Source. 8-12 week old female wild type CD26 positive (D⁺) and CD26negative (D⁻) Fischer rat (Charles River Labs, Japan) bone marrow.

Bone marrow preparation. Bone marrow was flushed from each femur, tibiaand fibula with Dulbecco's phosphate buffered saline (U-PBS) using a21-gauge needle and a 10 ml syringe. After removal, the bone marrow wasimmediately aspirated through the needle to make a single cellsuspension. The cells were washed twice with sterile PBS and resuspendedin MyeloCult H5100 long-term culture (LTC) media, available from StemCell Technologies, and supplemented with freshly diluted 10⁻⁶ Mhydrocortisone, available from Sigma.

Establishment of adherent bone marrow stromal cell cultures. 1-2×10⁷cells were seeded into a T25 flask (Corning) containing 10 mls of LTCmedium and incubated at 37° C. in 100% humidified 5% CO₂ in air. After 1week half the medium was exchanged for fresh medium and incubated forapproximately 1 week until a confluent cell layer formed.

PT-100 incubation. The adherent stromal cells were removed from theflask by trypsin-EDTA (Life Technologies, Inc.) digestion, washed oncewith sterile PBS and resuspended in long term culture medium at1×10⁵/ml. One ml of cells was seeded in each well of a 12-well tissueculture treated plate. PT-100 was diluted in medium and addedimmediately to the stromal cells at the appropriate concentrations (10⁻¹to 10⁻⁸ M) and the cultures were incubated at 37° C. in 100% humidified5% CO₂ in air.

Supernatant collection. The supernatants were collected from the stromalcultures after 1 or 2 days of incubation and either assayed immediatelyor stored at−20° C.

ELISA. The supernatants were assayed for IL-6 production using acommercially available ELISA kit (Biosource International).

DPPIV-like activity assay. Following the removal of supernatant from thestromal cultures, the adherent cells were washed with D-PBS and assayedfor DPPIV-like activity. The fluorogenic substrate Ala-Pro-AFC wasdiluted in DPPIV buffer (50 mM Hepes, 140 mM NaCl) from the originalstock of 10 mM (stored at−20° C. as a solution in dimethyl-formamide) to1 mM, directly before the assay. 30 μl of the 1 mM stock was added perml of buffer and the stromal cells were incubated for 10 minutes with 1ml per well of the diluted substrate. The reaction was stopped byremoving the substrate solution from each well and measured in thefluorescence spectrophotometer or stored−20° C. Activity was measuredusing an excitation wavelength of 400 nm and emission wavelength of 505nm.

Human Stromal Cultures

Cell source. Bone marrow was kindly donated by New England MedicalCenter, Boston, Mass. and Dr. Richard Benjamin from Brigham and Women'sHospital in Boston, Mass. Mononuclear cells were purified over Ficolland used immediately or frozen in liquid nitrogen.

Establishment of human stromal feeder layer. 2 to 4×10⁷ human bonemarrow mononuclear cells were seeded into a T75 flask (Corning)containing 20 mls of LTC medium and incubated at 37° C. in 100%humidified 5% CO₂ in air. After 1 week half the medium was exchanged forfresh medium and incubated for approximately 1 week when a confluentcell layer formed.

MyeloCult H5100 long-term culture (LTC) media can be obtained from StemCell Technologies and contains 12.5% horse serum, 12.5% fetal bovineserum, 0.2 mM I-inositol, 20 mM folic acid, 10⁻¹ 2-β-mercapto-ethanol, 2mM L-glutamine inα-MEM. Prior to using, add freshly diluted 10⁻⁶ Mhydrocortisone, available from Sigma.

ELISA of human cells. Cultured supernatants were assayed for a highsensitivity ELISA (R+D Systems).

Human cell surface staining. The phenotype of the established human bonemarrow stromal cells as determined using immunofluorescent staining withmonoclonal antibodies specific for CD26 and FAP alpha, followed byfluorescence activated cell sorting (FACscan, Becton Dickinson). FAPalpha was stained using a biotinylated monoclonal anti-human antibody(IgGl isotype) followed by Strepavidin-Fluorescein Isothiocyanin(SA-FITC). The antibody was purified over protein G, using culturedsupernatants from the hybridoma F19 (ATCC), and biotinylated usingbiotinyl-p-nitrophenyl ester (Sigma). CD26 was stained with Cy-chrome orPhycoerythrin anti-human CD26 (clone MA261). Mouse IgGl (Pharmigen) wasused for a negative control.

PT-100 incubation, supernatant collection and DPPIV-like activity assayfor human and rat cells were carried out in an identical manner.

Results:

Table 1 shows three representative experiments which demonstrate normalDPPIV-like activity on rat bone marrow stromal cells which lack CD26.The DPPIV-like activity is potently inhibited by PT-100. Bone marrowstromal cells were established from the long bones of Fischer D⁺ and D⁻rats and cultured for 2 days in the presence or absence of PT-100. Cellswere assayed for DPPIV-like activity as described in the Materials andMethods section. DPPIV-like activity is presented as velocity and thepercent of protease activity inhibition with PT-100 is calculatedrelative to a ‘no treatment’ control.

TABLE 1 Comparison of DPPIV-Like Activity of Fischer D⁺ and D⁻ Rat BMStromal Cells Experiment DPPIV-Like Activity % Inhibition by # RatPhenotype (Velocity = pMoles/min) 10⁻⁶ M PT-100 102098 D⁺ 23.4 76.9 D⁻23.3 62.1 102198 D⁺ 22.4 35.5 D⁻ 33.7 86.4 102398 D⁻ 35.8 73.8

FIG. 1 shows the comparison of the response of Fischer D+ and D− rat andbone marrow stromal cells to PT-100 in in vitro cultures. Similar levelsof IL-6 are secreted from bone marrow stromal cells of D⁺ and D⁻ rats.Moreover, IL-6 levels for both strains were enhanced by the addition ofPT-100. Bone marrow stromal cells were established from the long bonesof 3 Fischer D⁺ and D⁻ rats as described in Materials and Methods. Thestromal cells were incubated for 2 days in the presence or absence ofthe indicated concentrations of PT-100. IL-6 levels in the culturesupernatant were determined by ELISA.

A fluorescent activated cell sorting (FACS) profile of human bone marrowstromal cells is shown in FIG. 2. Human stromal cells can be establishedwhich lack cell surface CD26 but express the DPPIV-like protein FAP-α.Stromal cells were established from the bone marrow of a human volunteeras described in Materials and Methods. Cultured cells were removed bytrypsin digestion and assayed for surface expression of CD26 and FAP-αby immuno-fluorescence staining and cell sorting. The FACS profile forFAP-α (FL1) and CD26 (FL2) are represented as histograms. Negativecontrols used in this experiment were biotinylated mouse IgGl/SA-FITCand Cychrome-mouse IgGl (top panels). FAP alpha was stained withbiotinylated F19 followed by secondary staining with SA-FITC. CD26 wasstained with PE-mouse anti-human CD26.

FIG. 3 demonstrates that primary human stromal cells which express FAP,but not CD26, respond to PT-100. Human bone marrow cells that lack CD26but express FAP-α, as determined by FACS analysis, were potentlystimulated by PT-100 to release G-CSF. The DPPIV-like activityassociated with the intact cells was inhibited by PT-100. Stromal cellswere established from the bone marrow of a human volunteer as describedin the materials and methods. The stromal cells were incubated for 2days in the presence or absence of the indicated concentrations ofPT-100 and cultured supernatants were assayed for IL-6 or DPPIV-likeactivity as described in Materials and Methods.

Example 2

This example illustrates the anti-tumor activity of PT-100 (i.e.,Val-boro-Pro) in mouse models.

PT-100 administered orally significantly inhibits the growth of both theB16 melanoma and the WEHI-164 fibrosarcoma in mice.

The highly metastic B16-F0 tumor was implanted subcutaneously (s.c.) inC57BL/6 mice. PT-100 administration was started on day 8 when palpabletumors were apparent. Tumor volumes were measured on days 8, 12, and 15.The data are shown in FIG. 4. In mice treated with doses of 10 and 40 μgof PT-100 administered orally (p.o.), twice daily (b.i.d.) from day 8 to14, tumor growth was markedly inhibited compared with the saline treatedcontrol mice. A higher dose of 160 μg PT-100 administered for just twodays—day 8 and day 9—was also effective in suppressing tumor growth. Inthe experiment of FIG. 4 each experimental group contained 10 animals.

Similar experiments with the WEHI-164 fibrosarcoma demonstrated thatPT-100 could suppress the growth of an established s.c. tumor. Inaddition, when PT-100 administration was started shortly afterimplantation of WEHI-164 on day 2, it was found that not only was tumorgrowth inhibited, but a proportion of the tumors also became necroticand apparently regressed completely. Thus in the experiment of FIG. 5,the large reductions in tumor volume recorded were in part due to theabsence of a palpable tumor in some of the mice in each of the PT-100treated groups (10 replicate animals per group). For example, in themice treated with the 5 μg dose of PT-100 administered b.i.d., 6 of theanimals were completely free of a detectable s.c. tumor. PT-100treatment of the mice in the experimental groups was stopped on day 20,the day when the data of FIG. 5 were recorded. These mice have now beenmonitored up to day 30 after WEHI-164 implantation, and to date, none ofthe mice recorded as tumor-free on day 20 has developed a detectables.c. tumor.

Example 3

The in vivo effects of the compounds of the invention can be assayedthrough various animal models known to those of ordinary skill in theart. Generally such assays involve the injection of a carcinoma cellline, of mouse or preferably, human, origin, into a cohort of mice.Following the passage of several days, as determined by theproliferative rate of the cell line, parameters such as tumor size,degree of metastasis and cellular infiltration into a region in thevicinity of the tumor site are evaluated. In most experiments, twogroups of experimental mice are studied: a first, control, group whichreceives only the cell line but no agent of Formula I, and a second,test, group which receives at least one agent of Formula I such asPT-100. This second group is divided into several subgroups each ofwhich receives a different dose of the agent, preferably between 100 μgand 100 μg per day for 10-20 days. On these days, the control group willbe administered control doses containing only vehicle with no activeagent. The evaluation of tumor growth and organ specific metastasis willvary according to the tumor type studied.

In yet other assay systems aimed at determining the effect of the agentson metastatic spread or development, the agents are administered at thetime of inoculation of the malignant cells or short thereafter.Similarly, the agents may be administered after the innoculation, butbefore the full development of the tumor mass. In these ways, theeffects of agents on different stages of malignant growth and metastasiscan be tested.

The following are examples of different in vivo model systems forstudied a variety of epithelial cancers.

Lung carcinoma: One such example for lung carcinoma involves thesubcutaneous flank injection of the M109 mouse lung tumor cell line intosyngeneic mice. On each of days 4 through 8 after injection of the cellline, the mice receive single bolus daily doses of the compounds of theinvention by tail vein injection. The compounds of the invention areprepared and administered in a carrier solution which is physiologicallycompatible with both the recipient environment and the stability of thecompound. A preferable carrier solution is D-PBS with a carrier proteinsuch as albumin. Mice are sacrificed at day 4 after cell line injectionand at two day intervals after the administration of inhibitorycompounds. The tumors are excised and weighed. Measurement data can bestandardized relative to initial body weight of the recipient mouse.

Alternatively, if the transplantable tumor line is able to grow to theextent that it causes a reproducible and significant effect on totalmouse body weight, than the recipients need not be sacrificed. In thisreadout system, starting at day 0 (i.e., prior to the introduction ofthe cell line), the mice are weighed daily to determine the tumor burdenand to evaluate the effect of the injected compound(s) on tumor burden.Tumor mass can be calculated by the difference in mouse body mass duringthe experiment and at day 0. Measurement of control mice which receiveonly carrier solution with carrier protein will be used to standardizefor any unrelated weight gain.

Colon carcinoma: The effect of test agents on experimentally inducedhuman colorectal tumors in mice can be deduced by transplanting intonude mice human colon tumor cell lines such as COLO 205, C-1H, 26M3.1,CT-26, LS174T, and HT29, in a manner similar to that described above. Inthese models, pericecal tumor growth, angiogenesis, ascites andmetastasis to the liver are suitable readouts to ascertain if the testcompounds are active.

Melanoma and metastatic melanoma: Melanoma cell lines (e.g., B16 andSKMEL) are administered either intraperitoneally or intravenously ordirectly into the footpad. Primary tumor growth, survival time,resistance to tumor challenge, cellular infiltrates characteristic ofmelanoma tumors, and extent of tumor angiogenesis are all parameters ofinterest which can be evaluated. In certain models of melanoma,metastasis to the lung can be readily observed following surgicalremoval of the primary tumor.

Ovarian cancer: Human ovarian carcinoma cell lines such as JAM areadministered subcutaneously to severe combined immunodeficiency (SCID)mice. After 21 days, tumor growth is generally established and theeffects of the test agent after this point can be compared tovehicle-alone.

Breast cancer: Breast cancer cell lines such as MDA-MB-231 are injectedpreferably into the left cardiac ventricle of mice. Many breast cancersmetastasize to bone. About 4 weeks after inoculation, tumors and bonemetastases can be evaluated as can the effect of administration of thetest agent.

Squamous cell carcinoma: Human basaloid squamous cell carcinoma cells orestablished tumor lines such as HTB-1 are administered eithersubcutaneously or submucosally into mice. After allowing a sufficienttime for primary tumor growth, the mice are administered test or controlpreparations, and the effects of the test agent on the parametersdescribed above are determined.

Other transplantable cell lines useful in these assays include, but arenot limited to, human NCI-H522 lung tumor cell line (nude micerecipients), human SKOV3 cell line, and the M5076 cell line.

It should be understood that the preceding is merely a detaileddescription of certain preferred embodiments. It therefore should beapparent to those skilled in the art that various modifications andequivalents can be made without departing from the spirit and scope ofthe invention. It is intended to encompass all such modifications withinthe scope of the appended claims.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

1. A method for treating a subject having a condition characterized byan abnormal mammalian cell proliferation, comprising: administering to asubject in need of such treatment, an agent in an amount effective toinhibit the proliferation, wherein the agent is a compound of Formula I.2. The method of claim 1, wherein the abnormal mammalian cellproliferation is manifested as a tumor.
 3. The method of claim 1,wherein the condition is further characterized by the presence ofreactive stromal fibroblasts.
 4. The method of claim 1, wherein theabnormal mammalian cell proliferation is in epithelial cells.
 5. Themethod of claim 4, wherein the abnormal mammalian cell proliferation isselected from the group consisting of a carcinoma, a sarcoma, and amelanoma.
 6. The method of claim 1, wherein the condition is ametastasis of epithelial origin.
 7. The method of claim 1, wherein thecondition is selected from the group consisting of breast cancer,colorectal cancer, ovarian cancer prostate cancer, pancreatic cancer,kidney cancer, lung cancer, melanoma and fibrosarcoma.
 8. The method ofclaim 1, wherein the condition is selected from the group consisting ofbone and connective tissue sarcomas.
 9. The method of claim 1, whereinthe agent is administered locally.
 10. The method of claim 1, whereinthe agent is administered systemically.
 11. The method of claim 1,wherein the subject is otherwise free of symptoms calling forhemopoietic stimulation.
 12. The method of claim 1, wherein the agent isadministered in combination with surgery to remove an abnormalproliferative cell mass.
 13. The method of claim 1, wherein the agent isadministered to a patient who has had surgery to remove an abnormalproliferative cell mass.
 14. The method of claim 1, wherein the agent isadministered in combination with an anti-cancer compound.
 15. The methodof claim 1, wherein the agent is targeted to a tumor.
 16. The method ofclaim 1, wherein the subject has normal hemopoietic activity.
 17. Themethod of claim 1, wherein the subject is HIV negative.
 18. The methodof claim 1, wherein the agent is Val-boro-Pro.
 19. A method forinhibiting angiogenesis in a subject having a condition characterized byabnormal mammalian cell proliferation comprising: administering to asubject in need of such treatment, an agent in an amount effective toinhibit angiogenesis in an abnormal proliferative cell mass, wherein theagent is a compound of Formula I.
 20. The method of claim 19, whereinthe abnormal mammalian cell proliferation is manifested as a tumor. 21.The method of claim 19, wherein the abnormal mammalian cellproliferation is in epithelial cells.
 22. The method of claim 19,wherein the abnormal mammalian cell proliferation is selected from thegroup consisting of a carcinoma, a sarcoma, and a melanoma.
 23. Themethod of claim 19, wherein the condition is a metastasis.
 24. Themethod of claim 19, wherein the condition is selected from the groupconsisting of breast cancer, colorectal cancer, ovarian cancer, prostatecancer, pancreatic cancer, kidney cancer, lung cancer, melanoma andfibrosarcoma.
 25. The method of claim 19, wherein the agent isadministered locally.
 26. The method of claim 19, wherein the agent isadministered systemically.
 27. The method of claim 19, wherein thesubject is free of symptoms calling for hemopoietic stimulation.
 28. Themethod of claim 19, wherein the agent is administered in combinationwith surgery to remove an abnormal proliferative cell mass.
 29. Themethod of claim 19, wherein the agent is administered to a patient whohas had surgery to remove an abnormal proliferative cell mass.
 30. Themethod of claim 19, wherein the agent is administered in combinationwith an anti-cancer compound.
 31. The method of claim 19, wherein theagent is administered in combination with an anti-angiogenic compound.32. The method of claim 19, wherein the subject has normal hemopoieticactivity.
 33. The method of claim 19, wherein the subject is HIVnegative.
 34. The method of claim 19, wherein the agent is Val-boro-Pro.35. The method of claim 19, wherein the agent is targeted to a tumor.36. A pharmaceutical preparation comprising: an agent of Formula I, atleast one other anti-cancer compound, and a pharmaceutically acceptablecarrier.
 37. A pharmaceutical preparation comprising: an agent ofFormula I, at least one other anti-angiogenic compound, and apharmaceutically acceptable carrier.